The present invention relates to novel solid polymeric compositions, their preparation and cross-linking, and their uses.
A well-established method for improving the physical properties and solvent resistance of linear polymers is cross-linking, in which the individual polymer chains are joined at many points to yield an interconnected network. The cross-links may be ionic in character, as in complexes of poly-acids with poly-bases, but covalent or chemical cross-linking is stronger, more resistant to hydrolysis, and more versatile in its applications.
The most common way of achieving covalent cross-linking is to use a polyfunctional monomer in the polymerization reaction itself (e.g., divinylbenzene together with styrene). Since cross-linked polymers cannot be dissolved, melted or cast, the polymerization must be carried out in the final physical shape required. Problems often arise in the preparation of films and membranes when catalysts must be introduced or inhibitors such as oxygen excluded from the polymerization reaction. Furthermore, the monomers themselves are often too toxic, volatile, or fluid to be conveniently processed in this manner. All of these factors make the manufacturing process difficult and costly.
It is thus often preferably to prepare a linear polymer or polymers, dissolve them in an appropriate solvent, spray or cast in the final form required, and only then introduce the cross-links. A familiar example of such post-cross-linking is the vulcanization of rubber, in which linear polyisoprene is mixed with sulfur, molded and then heat-cured. Another example is the cross-linking of various unsaturated polymers by light to produce plates for photoengraving.
Although many polymers will form loosely cross-linked gels upon heating, the mechanism is often obscure and the cross-linking difficult to predict or control. The present invention provides a post-cross-linking method unique in its mechanism, in the range of polymers to which it applies, an in its practicality and wide range of application. It is believed to proceed via the alcoholysis of pendant amide or ester groups on the polymer with hydroxyl groups to produce a new ester linkage between polymer chains, or the amidolysis (or aminolysis) of pendant amide or ester groups on the polymer with amino groups to produce a new amide linkage between polymer chains, or the reaction of pendant carboxyl groups on the polymer with hydroxyl or amino groups to produce new ester or amid linkages. Depending on the nature of the reaction and the leaving group involved, the use of an acid or base catalyst or not catalyst at all is employed to obtain optimal cross-linking.
Alcoholysis or amides and esters is a well-known reaction in the organic chemistry of small molecules, where it generally is run in solution at reflux temperature with strong acid or base catalysts. It is one of several mechanisms postulated by Kopecek and Bazilova to account for unusually high molecular weights obtained in the solution polymerization of N-(2-hydroxypropyl) methacrylamide (European Polymer Jornal, 1973, vol. 9, pp. 10-11.) These authors considered only the possibility of dimerization, not actual cross-linking, and did not report any insolubilization taking place. The unusual feature of the alcoholysis, aminolysis and transamidation reactions described in this invention is their occurrence in the solid state in the absence of solvent, a phenomenon entirely unexpected and heretofore unreported.
Similarly, amidolysis of amides and esters is a well known reaction in the organic chemistry of small molecules, generally performed at elevated temperatures, sometimes with a base catalyst, other times with an acid catalyst or no catalyst at all depending on the nature of the solvent and the leaving group. The novel post-cross-linking techniques taught by this invention have many unique advantages, particularly in the manufacture of synthetic membranes. Conventional membranes for reverse osmosis (RO) and ultrafiltration (UF) are made of polymers that are insoluble in the fluid acted on by the membrane (water, in most cases). Typically, a linear polyamide, polysulfone, or cellulose acetate is cast from an organic solvent and coagulated in water. Although such membranes are rigid and physically strong, they are hydrophobic in nature and tend to foul through adsorption of hydrophobic particles and solutes in the feed stream. Such fouling is a major problem in industrial use of membranes, making frequent cleaning or costly pretreatment necessary.
Strongly hydrophilic polymers, particular those where in at least 80% of their constituent units are highly polar so the linear polymer is highly soluble in water, which include those having a fixed positive or negative charge on each unit or an uncharged species with a similarly high affinity for water such as acrylamide, have been shown to resist such adsorptive fouling. However, they either dissolve in water or, unless substantially cross-linked, form a soft gel. Useful membranes may be formed of such polymers only by a high degree of cross-linking, so they swell not more than 3-5 times by weight when soaked in water. Since the pore structure of such membranes is generally created by coagulation and since monomers are essentially uncoagulable, the best practical route is post-cross-linking of the coagulated linear polymer. Using the techniques taught by this invention, highly cross-linked UF membranes of controlled porosity may be cast from very hydrophilic polymers. The intricate pore structure of these coagulated membranes is preserved by solid state post-cross-linking. Furthermore, charged functionalities such as sulfonate or quaternary ammonium may be incorporated to yield a cross-linked interpolymer membrane of the type described by Gregor (U.S. Pat. N. 3,808,305), where the fixed charges serve to reject charged colloidal particles and, to a lesser extent, dissolved salts.
A typical and commonly employed hydrophilic support medium is cross-linked polyacrylamide (PAM) beads of different sizes and different degrees of cross-linking, the latter acting to control pore size. PAM contains a hydrocarbon skeletion to which are attached pendant amidocarbonyl groups which contain an amide ammonia nitrogen, one readily replaced by certain other nitrogen compounds, thus permitting the formation of several derivatives having useful properties. A typical, earlier study by Inman and Dintzis (Biochemistry, 1969, vol. 8, pp. 4074-4082.) showed that commercially available beads made of cross-linked copolymers of acrylamide and N, N'-methylenebisacrylamide could be treated by several different reactions to make useful products. These authors performed the direct aminoethylation of the beads as well as the preparation of a number of derivatives from these beads including those of the: hydrazide; trinitrophenyl; 2,4 dinitrosophenylaminoethyl; succinylhydrazide; sulfoethyl; p-hydroxyphenethyl. Also, these authors found it was possible to effect the coupling of primary amines via the general acyl azide procedure employing, in this case, the hydrazide derivative. These authors also achieved coupling to proteins such as to serum albumin via the acyl azide reaction, and to trypsin via the treatment of the hydrazide derivative with cold nitrous acid, followed by treatment with a suitable solution of trypsin. These authors also converted the aminoethyl derivative of PAM with p-nitrobenzoylazide, followed by triethylamine and washing with DMF to form the p-nitrobenzamidoethyl derivative which was then converted to the p-aminobenzamidoethyl derivative. The latter, after treatment with nitrous acid was employed for the coupling of bovine serum albumin via the diazonium intermediate. These authors then employed some of these derivatives as immobilized enzymes and as immunoadsorbents.
The materials which can be prepared by the teachings of the subject invention constitute, particularly useful and powerful tools for the techniques of modern biochemistry and molecular biology. Often these require a sequential and laborious laboratory procedure which usually must be carried out over a period of several days. Often one starts with substantial quantities of material, but very soon this is reduced to a dilute solution containing a small amount of material, of which a very small fraction is the desired component. The classical biochemical techniques which involve salt precipitation, desalting and successive column chromatography, with a final concentration often achieved by freeze-drying and subsequent procedures, results in both an extremely laborious and time-consuming procedure and usually a loss or denaturization of the very substance which is desired as an end product of the entire process.
As a typical example, in the isolation of transcription factors, one first usually uses a large column which can be cross-linked heparin, .alpha.hydroxy-apetite or a phosphocellulose, for the purpose of concentrating the DNA binding proteins which have an affinity for these columns. The next step is to elute the active material, usually by the use of different salt gradients and test each eluted fraction for specific activity. Once an active fraction is isolated, it can be separated by using gel permeation chromatography to visualise the amount and approximate molecular weight of the protein in that fraction, and finally a certain band is isolated which contains substantial amounts of the activity of the proteins which constitute a transcription factor. Since none of these preparatory procedures produces a single band of the desired, purified protein, in virtually all circumstances the final purification procedure required consists of growing antibodies to the protein desired and employing these antibodies in an affinity chromatography system to finally obtain the pure protein. Since a desired component is a low molecular weight material such as a polypeptide present in a group of highly similar polypeptides, the problem is a particularly difficult one.
All of these procedures, involving as they do for the most part hydrophobic materials or surfaces to which the desired proteins may be adsorbed and lost, suffer from that disadvantage.